The long range goal of this project still remains to understand the role that polyadenylation plays in posttranscriptional regulation in prokaryotes. Although long considered to be a feature unique to eukaryotes, over the past 10 years it has been shown that polyadenylation is intimately involved in mRNA decay, rRNA processing, tRNA maturation and overall RNA quality contol in Escherichia coli. Of equal importance, it has now been shown that polyadenylation, either by poly(A) polymerase type enzymes or polynucleotide phosphorylase are involved in the post-transcriptional modification in almost all prokaryotes. In addition, over the past few years it has been shown that eukaryotes contain a nuclear polyadenylation system that closely mimics the E. coli system in that it targets defective Pol II transcripts for degradation. Thus E. coli is an excellent prokaryotic model system for studying this complex system. Based on the progress that we have made during the current grant period, we are now in a position to develop a much more sophisticated understanding of both the biochemical mechanism of polyadenylation and the roles it plays in mRNA decay, tRNA maturation and rRNA processing. Specific experimental approaches include: 1. Determine whether PAP I preferentially polyadenylates full-length or partially degraded transcripts; 2. Determine how polyadenylation helps initiate mRNA decay; 3. Analysis of the polyadenylation complex at physiological levels of PAP I; 4. Characterization of the polyadenylation complex in vitro; 5. Identification and characterization of the stationary phase-specific poly(A) polymerase; 6. Determine the essential features that make a Rho-independent transcription terminator a polyadenylation signal; 7. Analyze the posttranscriptional modification of polycistronic transcripts terminating with or without a Rho-independent transcription terminator; 8. Determine the role of polyadenylation in the maturation of rRNAs and tRNAs; and 9. Analyze the interactions between poly(A) tails and the RNase E based degradosome in the initiation of mRNA decay. Since polyadenylation appears to be present in a wide range of prokaryotes, including many pathogenic organisms, a more complete understanding of this system provides an opportunity to identify new targets for anti-microbials that will be active against either gram negative or gram positive bacteria.